PROJECT SUMMARY Once rapid embryonic and neonatal cellular expansion is completed, hematopoietic stem cells (HSC) withdraw from the cell cycle, and serve as a reservoir to sustain the production of all blood cells throughout adult life. HSCs are functionally heterogenous and contain cells with disparate differentiation and durable engraftment potential. However, molecular drivers of adult HSC remain enigmatic. We have developed several mouse models and deep genomics data sets which support the existence of discrete HSC cell states that differ both molecularly and functionally. Moreover, we find that HSC history of division may account for these genomic differences; placing these populations in a hierarchical structure based on divisional history. Further, our data suggest that HSC dramatically remodel the mitochondrial network upon entry into cell cycle and that mitochondria do not return to a homeostatic state after returning to quiescence. We hypothesize that HSC are hierarchically organized in functionally distinct HSC states, and that this organization can be resolved by their divisional history for which mitochondria provide memory. The proposed work will first incisively establish the molecular architecture of discrete HSC states, including drivers of the most functional population, then define the role of mitochondria in functional programming of discrete HSC populations, including how alterations in mitochondria maintenance contribute to a decline in fitness. The overarching goal is to define the cell states encountered by HSC and their derivatives, as well as to provide mechanistic insight into the underlying transcriptional circuits and cell biological changes indicative of transition between states. We expect the proposed research to contribute to a fundamental understanding of the hematopoietic system ? information that can be used to develop new modalities for HSC expansion, validate grafts before BMT, or safely genetically manipulate HSC for gene therapy.